Vertebrate bone is a dynamic tissue whose mass can change in response to an individual's age, calcium homeostatic demands and physiological conditioning. Bone mass changes are the result of coordinated contrasting activities of resident osteoblasts, responsible for bone deposition, and osteoclasts, that resorb bone and promote loss of the tissue. Controlling mechanisms for osteoblast and osteoclast activity include blood calcium titre and hormonal regulators, particularly the gonadal steroids such as estrogen, and peptide hormones including calcitonin (CT). Postmenopausal women experiencing severe estrogen deficiency, often suffer enhanced osteoporotic bone loss, which may be arrested with estrogen replacement therapy and although controversial, many studies have measured a postmenopausal decrease of plasma immunoreactive CT. Because CT is known to inhibit osteoclast activity, a deficiency of CT may result in increased bone resorption contributing to osteoporosis; CT therapy alone can limit osteoporotic bone loss. Although studies have demonstrated elevated plasma CT following estrogen replacement therapy, and an estrogen induction of CT secretion in vitro, little is known of specific molecular mechanisms that account for the relationship between estrogen and CT. An invertebrate model for the study of steroid and CT interactions and their regulation of calcified tissue dynamics exists in the crustacean arthropods. Members of this group of organisms possess a calcified cuticle, or exoskeleton, composed of both organic matrix, and inorganic mineralized calcium in close proportions to that of vertebrate bone. The exoskeleton is periodically removed during molting (ecdysis), a process that includes prior calcium and matrix resorption, and must then be resynthesized by rapid biomineralization and organic matrix secretion. The molting process is controlled by ecdysteroids whose actions are mediated by nuclear receptors that have recently been cloned and found to be structurally and functionally analogous to other steroid hormone receptors. Reportedly, CT is not only present in crustaceans, but its levels are highest immediately following the peak of ecdysteroids preceding ecdysis. The applicants have reported the identification of crustacean CT mRNA in crayfish. Considering that crustaceans resorb and synthesize a calcified matrix by a steroid- regulated process, and that CT is also present and regulated during the molt cycle, these organisms will serve as excellent models for the study of steroid-CT interactions. The Specific Aims of this proposal are to: (1) use crayfish to characterize the steroid (ecdysteroid) regulation of CT; and (2) investigate the possible molecular mechanism for ecdysteroid control of CT by examination of the crayfish CT gene promoter for ecdysteroid response elements (EcREs). Both in vivo and in vitro experiments will be used to establish the temporal control of ecdysteroid on crayfish CT synthesis and secretion. A functional analysis using reporter gene constructs and transfection of ecdysteroid-responsible cultured cells, will be used to reveal the presence and structure of EcREs in the crayfish CT promoter. The detailed study of exogenous steroid on Ct- producing tissue and the examination of the CT gene for functional steroid REs should contribute much to the elucidation of the relationship between steroid hormones and CT in vertebrates and the development of osteoporosis therapies.